PROJECT SUMMARY/ABSTRACT Lung cancer is a prevalent disease and consumes many lives every year. Cancer immunotherapy using immune checkpoint inhibitors (ICIs; e.g. anti-PD-1antibody) has revolutionized the treatment of metastatic lung cancer, resulting in long-term complete responses for many patients. Nevertheless, there remains an urgent need for new strategies because not all patients respond to ICIs; moreover, resistance can occur in those that do. Myeloid-derived suppressor cells (MDSCs), in particular, monocytic MDSCs (M-MDSCs) are potent immunosuppressive innate immune cells that actively inhibit CD8+ T cell tumor homing and activation. Since M-MDSC levels are elevated in multiple human cancers and correlate with decreased patient survival, we postulate that these cells contribute to anti-PD-1 resistance. The purine nucleoside, adenosine, is produced in copious amounts within the tumor microenvironment (TME), where it serves to suppress the immune system and promote tumor growth. There is evidence to suggest that overproduction of adenosine can mediate resistance to ICIs. Immune suppressive adenosine is produced via the enzymatic conversion of extracellular AMP by the cell surface enzyme, CD73 (ecto-5?-nucleotidase; AMP ? adenosine). Our preliminary studies demonstrate that tumor cell-derived prostaglandin E2 (PGE2) maintains M-MDSC suppressive activity, in large part, by directly inducing cell surface CD73 expression leading to increased immune suppressive adenosine within the TME. The overall hypothesis of this proposal is that tumor cell-derived PGE2 dictates CD73 expression in M-MDSCs leading to substantial increases in adenosine-dependent inhibition of anti-tumor CD8+ T cell activation resulting, ultimately, in anti-PD-1 immunotherapeutic resistance. A major goal of this proposal is to test the anti-tumor efficacy of a novel cancer immunotherapy involving systemic administration of adenosine deaminase (ADA)?an enzyme that irreversibly converts adenosine into inosine, a non- immunosuppresive nucleoside. A pegylated version of bovine ADA (PEG-ADA) is already FDA-approved for use as an enzyme replacement therapy in children with severe combined immunodeficiency (ADA-SCID). We hypothesize that depletion of adenosine-mediated T cell immune suppression by PEG-ADA sensitizes tumors to PD-1 inhibitor therapy and improves clinical outcomes for NSCLC patients. To fulfill the stated objectives, the following aims are proposed: 1) Investigate intra-tumoral M-MDSC functional maintenance in the context of a PGE2 ? CD73+ M-MDSC ? adenosine dependent pathway; 2) Determine if loss/inhibition of extracellular adenosine-dependent pathway attenuates anti-PD-1 resistance in mouse models of lung cancer; and 3) Validate PGE2 ? CD73+ M-MDSC ? adenosine mediated anti-PD-1 resistance pathway in lung cancer patients receiving pembrolizumab therapy. Our proposed study will provide important insights towards developing a safe and novel immunotherapy to attenuate ICI resistance in lung cancer patients.